Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for the production of recombinant proteins on a commercial scale.
The biotech industry makes an extensive use of mammalian cells for the manufacturing of recombinant glycoproteins for human therapy.
Suitable cells that are widely used for production of polypeptides turned out to be Chinese Hamster Ovary (CHO) cells.
CHO cells were first cultured by Puck (1958) from a biopsy of an ovary from a female Chinese hamster. From these original cells a number of sub-lines were prepared with various characteristics. One of these CHO cell lines, CHO-K1, is proline-requiring and is diploid for the dihydrofolate reductase (DHFR) gene. Another cell line derived from this cell line is a DHFR deficient CHO cell line (CHO DUK B11) (PNAS 77, 1980, 4216-4220), which is characterized by the loss of DHFR function as a consequence of a mutation in one DHFR gene and the subsequent loss of the other gene.
Further cells that are frequently used for the production of proteins intended for administration to humans are human cell lines such as the human fibrosarcoma cell line HT1080 or the human embryonic kidney cell line 293, a human embryonic retinoblast-derived cell line such as e.g. PER.C6, an amniotic cell derived-cell line or a neuronal-derived cell line.
Cells from a suitable cell line are stably transfected with an expression vector comprising the coding sequence of the protein of interest to be produced, together with regulatory sequences such as promoters, enhancers, or polyA signals that ensure stable and correct expression of the protein of interest. Further genes usually present on expression vectors are marker genes such as e.g. positive selections markers (e.g. neo gene) that select the stably transfected cells from the untransfected and transiently transfected cells. Amplifiable genes such as the DHFR gene are used for amplification of the coding sequences.
Once a clone expressing the protein of interest has been established, a manufacturing process starting from this clone must be established allowing for production in high amounts and such a quality as is required for proteins destined for human administration.
Such manufacturing processes are generally carried out in bioreactors. There are different modes of operation. Today, fed-batch and perfusion cultures are the two dominant modes of industrial operation for the mammalian cell culture processes that require large amount of proteins (Hu and Aunins 1997). Whatever the production technology of choice is, development efforts aim at obtaining production processes that warrant high volumetric productivity, batch-to-batch consistency, homogenous product quality at low costs.
The decision between fed-batch or perfusion production mode is mainly dictated by the biology of the clone and the property of the product, and is done on a case-by-case basis during the course of the development of a new drug product (Kadouri and Spier 1997).
When the selection is a perfusion process, one of the culture systems of choice is stationary packed-bed bioreactor in which cells are immobilized onto solid carriers. This system is easy to operate and with appropriate carriers and culture conditions very high cell density (of ˜107-108 cell·ml−1) can be achieved.
A consequence of this high cell density is the need for an intensive medium perfusion rate (feed and harvest) that should be used in order to keep the cells viable and productive. It appears that the perfusion rate is one of the central parameters of such a process: it drives the volumetric protein productivity, the protein product quality and has a very strong impact on the overall economics of the process.
For the cell culture process, in the past culture media were supplemented with serum, which serves as a universal nutrient for the growth and maintenance of all mammalian cell lines that produce biologically active products. Serum contains hormones, growth factors, carrier proteins, attachment and spreading factors, nutrients, trace elements, etc. Culture media usually contained up to about 10% of animal serum, such as fetal bovine serum (FBS), also called fetal calf serum (FCS).
Although widely used, serum has many limitations. It contains high levels of numerous proteins interfering with the limited quantities of the desired protein of interest produced by the cells. These proteins derived from the serum must be separated from the product during downstream processing such as purification of the protein of interest, which complicates the process and increases the cost.
The advent of BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period, has raised regulatory concerns about using animal-derived sera in the production of biologically active products.
There is therefore a great demand for the development of alternative cell culture media free from animal sources that support cell growth and maintain cells during the production of biologically active products.
Generally, cell culture media comprise many components of different categories, such as amino acids, vitamins, salts, fatty acids, and further compounds:                Amino acids: For instance, U.S. Pat. No. 6,048,728 (Inlow et al.) discloses that the following amino acids may be used in a cell culture medium: Alanine, Arginine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamin, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenyalanine, Proline, Serine, Tryptophan, Tyrosine, Threonine, and Valine.        Vitamins: US 2003/0096414 (Ciccarone et al.) or U.S. Pat. No. 5,811,299 (Renner et al.) for example describe that the following vitamins may be used in a cell culture medium: Biotin, Pantothenate, Choline Chloride, Folic Acid, Myo-Inositol, Niacinamide, Pyridoxine, Riboflavin, Vitamin B12, Thiamine, Putrescine.        Salts: For instance, U.S. Pat. No. 6,399,381 (Blum et al.) discloses a medium comprising CaCl2, KCl, MgCl2, NaCl, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic, Sodium Selenite, CuSO4, ZnCl2. Another example for a document disclosing the inorganic salts that may be used in a culture medium is US 2003/0153042 (Arnold et al.), describing a medium comprising CaCl2, KCl, MgCl2, NaCl, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic, CuCl2.2H2O, ZnCl2.        Fatty acids: Fatty acids that are known to be used in media are Arachidonic Acid, Linoleic Acid, Oleic Acid, Lauric Acid, Myristic Acid, as well as Methyl-beta-Cyclodextrin, see e.g. U.S. Pat. No. 5,045,468 (Darter). It should be noted that cyclodextrin is not a lipid per se, but has the ability to form a complex with lipids and is thus used to solubilize lipids in the cell culture medium.        Further components, in particular used in the frame of serum-free cell culture media, are compounds such as glucose, glutamine, Na-pyruvate, insulin or ethanolamine (e.g. EP 274 445), or a protective agent such as Pluronic F68. Pluronic® F68 (also known as Poloxamer 188) is a block copolymer of ethylene oxide (EO) and propylene oxide (PO).        
Standard “basic media” are also known to the person skilled in the art. These media already contain several of the medium components mentioned above. Examples of such media that are widely applied are Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), or Ham's medium.
After production of the protein of interest in the bioreactor, the protein of interest needs to be purified from the cell culture harvest. The cell culture harvest may e.g. be cell extracts for intracellular proteins, or cell culture supernatant for secreted proteins.
While many methods are now available for large-scale preparation of proteins, crude products, such as cell culture harvest, contain not only the desired product but also impurities which are difficult to separate from the desired product.
The health authorities request high standards of purity for proteins intended for human administration. As a further difficulty, many purification methods may contain steps requiring application of low or high pH, high salt concentrations or other extreme conditions that may jeopardize the biological activity of a given protein. Thus, for any protein it is a challenge to establish a purification process allowing for sufficient purity while retaining the biological activity of the protein.
Ion exchange chromatographic systems have been used widely for separation of proteins primarily on the basis of differences in charge. In ion exchange chromatography, charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). Resins that may be used in ion exchange chromatography may contain different functional groups: diethylaminoethyl (DEAE) or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, while carboxymethyl (CM) and sulphopropyl (SP) have sodium as counter ion, for example.
Chromatographic systems having a hydrophobic stationary phase offer an alternative basis for separations and have also been widely employed in the purification of proteins. Included in this category are hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC). The physicochemical basis for separation by HIC and RPLC is the hydrophobic effect, proteins are separated on a hydrophobic stationary phase based on differences in hydrophobicity.
Reverse phase chromatography is a protein purification method closely related to HIC, as both are based upon interactions between solvent-accessible non-polar groups on the surface of biomolecules and hydrophobic ligands of the matrix. However, ligands used in reverse phase chromatography are more highly substituted with hydrophobic ligands than HIC ligands. While the degree of substitution of HIC adsorbents may be in the range of 10-50 μmoles/mL of matrix of C2-C8 aryl ligands, several hundred μmoles/mL of matrix of C4-C8 alkyl ligands are usually used for reverse phase chromatography adsorbents.
The Source 30RPC column is a polymeric reverse phase matrix. It is based on rigid, monosized 30 micron diameter polystyrene/divinyl benzene beads. Its characteristics can be summarized as follows: Exceptionally wide pH range (1-12), high selectivity, high chemical resistance, high capacity and high resolution at high flow rates.
Size-exclusion chromatography (SEC), also called gel-permeation chromatography (GPC), uses porous particles to separate molecules of different sizes. It is generally used to separate biological molecules and to determine molecular weights and molecular weight distributions of polymers. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transit time than larger molecules that cannot enter the particles. All molecules larger than the pore size are not retained and elute together. Molecules that can enter the pores will have an average residence time in the particles that depends on the molecules size and shape. Different molecules therefore have different total transit times through the column.
Blue Sepharose is a chromatography resin based on a dye-ligand affinity matrix. The ligand, Cibacron Blue F3G-A, is covalently coupled to Sepharose™ through chlorotriazine ring (Clonis et al., 1987).
Blue Sepharose has been used for the purification of interferon beta (Mory et al., 1981).
Interferon beta (interferon-β or IFN-β) is a naturally occurring soluble glycoprotein belonging to the class of cytokines. Interferons (IFNs) have a wide range of biological activities, such as anti-viral, anti-proliferative and immunomodulatory properties.
The three major interferons are referred to as IFN-alpha, IFN-beta and IFN-gamma. These interferons were initially classified according to their cells of origin (leukocytes, fibroblasts or T-cells). However, it became clear that several types might be produced by one cell. Hence leukocyte interferon is now called IFN-alpha, fibroblast interferon is IFN-beta, and T-cell interferon is IFN-gamma. There is also a fourth type of interferon, lymphoblastoid IFN, produced in the “Namalwa” cell line (derived from Burkitt's lymphoma), which seems to produce a mixture of both leukocyte and fibroblast IFN.
Human fibroblast interferon (IFN-beta) has antiviral activity and is also known to inhibit proliferation of cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs. From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology, Derynck et al., 1980 deduced the complete amino acid sequence of the protein, which is 166 amino acid long.
Interferon-β has also been cloned. U.S. Pat. No. 5,326,859 describes the DNA sequence of human IFN-β and a plasmid for its recombinant expression in bacteria such as E. coli. European Patent No. 0 287 075 describes a CHO (Chinese Hamster Ovary) cell line, transfected with the interferon-β coding sequence and capable of producing recombinant interferon-β. The protein is described as being glycosylated with a biantennary (two branched) oligosaccharide, featuring a single fucose moiety.
Interferon beta has been expressed in several cell lines, such as CHO cells, BHK 21 (baby hamster kidney cells) and LTK (mouse L-thymidine kinase negative) cells (Reiser and Hauser, 1987). DHFR negative CHO cells have also been used for the expression of interferon beta (Innis and McCormick, 1982), (Chernajovsky et al., 1984).
Interferons are known to be glycosylated, often with different glycoforms. For example, the saccharide structure of IFN-β was shown to include a bi-antennary structure, featuring a single fucose saccharide and terminal galactose sialylation (Conradt et al., 1987). Glycosylation was shown to also be important for solubility, since the IFN-β precipitated after deglycosylation with glycopeptidase F. In addition, IFN-β produced by E. coli showed folding problems, due to lack of glycosylation in the bacterial expression system.
European Patent No. 0 529 300 describes a recombinant interferon-β having a specific glycosylation pattern, namely glycosylation with carbohydrate structures that feature one fucose per oligosaccharide unit. These carbohydrate structures are biantennary, triantennary and tetraantennary (two, three and four branched, respectively) oligosaccharides.
PCT Application No. WO 99/15193 also describes glycosylation of recombinant interferon-β featuring biantennary, triantennary and tetraantennary oligosaccharides. The constituent monosaccharides included mannose, fucose, N-acetylglucosamine, galactose and sialic acid.
Various studies have demonstrated the importance of glycosylation for stability. For example, non-glycosylated forms of recombinant interferon-β were shown to have significantly lower stability and also lower biological activity (Runkel et al., 1998).
Other studies have shown that recombinant and natural human interferon-β proteins have different glycosylation patterns (Kagawa et al., 1988).
Interferon beta is used as a therapeutic protein drug, a so-called biological, in a number of diseases, such as e.g. multiple sclerosis, cancer, or viral diseases such as e.g. SARS or hepatitis C virus infections.
Therefore, there is a need for processes for the efficient production and purification of interferon beta, and of cells expressing interferon beta in high amounts.